Attenuation of Late-Stage Disease in Mice Infected bythe Mycobacterium tuberculosis Mutant Lacking theSigF Alternate Sigma Factor and Identification ofSigF-Dependent Genes by MicroarrayAnalysis

Abstract

The Mycobacterium tuberculosis alternate sigma factor, SigF, is expressed during stationary growth phase and under stress conditions in vitro. To better understand the function of SigF we studied the phenotype of the M. tuberculosis ⌬sigF mutant in vivo during mouse infection, tested the mutant as a vaccine in rabbits, and evaluated the mutant's microarray expression profile in comparison with the wild type. In mice the growth rates of the ⌬sigF mutant and wild-type strains were nearly identical during the first 8 weeks after infection. At 8 weeks, the ⌬sigF mutant persisted in the lung, while the wild type continued growing through 20 weeks. Histopathological analysis showed that both wild-type and mutant strains had similar degrees of interstitial and granulomatous inflammation during the first 12 weeks of infection. However, from 12 to 20 weeks the mutant strain showed smaller and fewer lesions and less inflammation in the lungs and spleen. Intradermal vaccination of rabbits with the M. tuberculosis ⌬sigF strain, followed by aerosol challenge, resulted in fewer tubercles than did intradermal M. bovis BCG vaccination. Complete genomic microarray analysis revealed that 187 genes were relatively underexpressed in the absence of SigF in early stationary phase, 277 in late stationary phase, and only 38 genes in exponential growth phase. Numerous regulatory genes and those involved in cell envelope synthesis were down-regulated in the absence of SigF; moreover, the ⌬sigF mutant strain lacked neutral red staining, suggesting a reduction in the expression of envelope-associated sulfolipids. Examination of 5-untranslated sequences among the downregulated genes revealed multiple instances of a putative SigF consensus recognition sequence: GGTTTCX 18 GGGTAT. These results indicate that in the mouse the M. tuberculosis ⌬sigF mutant strain persists in the lung but at lower bacterial burdens than wild type and is attenuated by histopathologic assessment. Microarray analysis has identified SigF-dependent genes and a putative SigF consensus recognition site.

Global transcriptional response to vancomycin in Mycobacterium tuberculosis

“…It was one of the genes with the largest reduction in transcription during early stationaryphase growth of a M. tuberculosis mutant harbouring a deletion of the gene encoding s F (Geiman et al, 2004), and it has been shown to interact physically with this sigma factor in a two-hybrid system (Parida et al, 2005). Its coinduction with s E opens the interesting hypothesis that this protein could be involved in the regulation of the activity of this sigma factor.…”

Paradoxical conservation of a set of three cellulose-targeting genes in Mycobacterium tuberculosis complex organisms

“…The transcription of Cel6 was predictable, as this gene has been cloned and the encoded protein is expressed in Escherichia coli in an active form (Varrot et al, 2005). Moreover, the Cel6 gene has been shown to be downregulated in an M. tuberculosis mutant lacking SigF (Geiman et al, 2004), indicating that the regulation of this gene is SigF-dependent. The Cel12 gene lacks a peptide signal and exhibits a potentially fragmented structure which could suggest gene decay.…”

The alternative sigma factor SigF of Mycobacterium smegmatis is required for survival of heat shock, acidic pH and oxidative stress

“…While none of the mutants was impaired in growth in broth culture or human macrophages in vitro, they showed reduced virulence in mouse and guinea pig infection models (Chen et al, 2000;Geiman et al, 2004;Karls et al, 2006). Furthermore, it was shown that SigF was required for survival of M. tuberculosis in the lung, but not in the spleen of guinea pigs (Karls et al, 2006).…”

“…No induction compared to unexposed control cells was observed under these conditions (data not shown), confirming that the observed induction of rsbW-lacZ was indeed due to exposure to D-cycloserine. In M. tuberculosis, SigF appears to have a role in the regulation of genes involved in cell envelope biosynthesis (Geiman et al, 2004;Williams et al, 2007). It may therefore be that cell wall stress, e.g.…”